Dynamic Changes of Mouse Embryonic Stem Cell-Derived Neural Stem Cells Under In Vitro Prolonged Culture and Hypoxic Conditions

Embryonic stem (ES) cells have been utilized as an excellent model for the study of neural development. However, the dynamic changes of ES cell-derived neural stem cells (ES-NSCs), under the effects of prolonged cell culture and hypoxic conditions, are still obscured. In the present study, using the combination of serum-free culture of embryoid body-like aggregates (SFEB) culture and cell sorting by Sox-1, the ES-NSCs were easily isolated and showed in vitro temporal neural specification, which resulted in distinct cell fates after neural differentiation. Early passaged ES-NSCs gave rise to neurons, whereas late-passaged ES-NSCs gave rise to glial cells, similar to the in vivo dynamic changes during the neural development. Remarkably, hypoxia treatment induced the neural differentiation of ES-NSCs but did not affect the cell fate. Under hypoxic conditions, early passaged ES-NSCs showed the upregulation of neuronal markers, whereas late-passaged ES-NSCs showed the upregulation of a glial marker. In addition, the knockdown of the hypoxia-inducible factor 1α expression impaired the neuronal differentiation of early passaged ES-NSCs under hypoxic conditions. These data demonstrated the distinct effects of prolonged culture and hypoxic stimuli on the neural differentiation of ES-NSCs; prolonged culture was involved in the cell fate after neural differentiation, while hypoxia treatment efficiently promoted neural differentiation.